Leishmania donovani persistence and circulation causing cutaneous leishmaniasis in unusual-foci of Nepal

Cutaneous leishmaniasis cases have increased dramatically in recent years in Nepal. The study offers molecular identification of the Leishmania species using 40 patient’s aspiration biopsy samples, targeting markers kinetoplast minicircle DNA (kDNA) and internal transcribed spacer-1 (ITS1). Among molecularly diagnosed 22 cutaneous leishmaniasis cases, L. donovani complex was identified in 13 instances and L. major in 9 cases. The ITS1 PCR was positive in 12 of the positive nested- kDNA PCR cases (12/22), confirming L. donovani complex in seven of the cases and L. major in five of the cases. In addition, the study conclude that concurrent occurrence of atypical cutaneous infections caused by L. donovani parasite in 59.1% of cases and typical cutaneous infections caused by L. major parasite in 40.9% of cases. A Phylogentic analaysis showed that the detected L. donovani species present null genetic distances from seven references of L. donovani, but slight differences between ITS1 sequences and not grouped into a significant monophyletic cluster.


Results
Demographic and geographic distribution of cutaneous leishmaniasis cases. In this study, interestingly a good numbers of suspected cases (n = 40) visited the hospitals on their own initiative in two-year study period. Among them, 65% (n = 26) were males and 35% (n = 14) were females. Among 22 (55%) cutaneous leishmaniasis cases as confirmed by this study using parasitological and/or molecular diagnosis, genderwise disribution showed 59.09% (n = 13) male while 40.9% (n = 9) were female. The research data showed that highest (n = 7) number positive cutaneous leishmaniasis cases fell into age group ≤ 20 years and in 41-60 years age group while the lowest (n = 1) number fell into age groups > 80 years (Fig. 1).
Cutaneous leishmaniasis cases were detected from all the geographical landscapes of Terai (latitude 26°), hill (latitude 27°-29°) and mountain (latitude 29°) regions. The coverage of cutaneous leishmaniasis cases was seen in all provinces encompassing 16 districts during the period of this study (Table 1, Fig. 2). The highest number of positive cutaneous leishmaniasis cases were found in hill (n = 13) followed by mountain (n = 7) and least in the Terai (n = 2) region covering 11, 3 and 2 districts of Nepal respectively. Similarly, Province Gandaki (Province 4), Province Karnali (Province 6) and Province Sudur Pashchim (Province 7) had equal five cutaneous leishmaniasis cases. Among the two districts recorded with prevalence in Province 7, Bajura district had three cases and Baitadi district had two cases. In Province 4; Gorkha, Tanahun, Baglung and Syangja districts had total five positive cases. In province 6, three cases were from the Kalikot district, and two other cases were from Jajarkot and Humla districts. There were two cases in Province Lumbini (Province 5) from Palpa and Rukum districts. In Province Koshi (Province 1), three cases were from Jhapa, Bhojpur and Okhaldhunga districts and only one case in each Province Bagmati (Province 3) and Province Madhesh (Province 2) from Ramechhap and Rautahat districts respectively (Table 1). In reference to national and international travel history of the patients, only five patients reported their travel to different countries, namely Saudi Arabia, United Arab Emirates (Dubai), India and UK, while only five patients reported their travel to another district or province than his place of residence (Supplementary Table S-II).
Clinical manifestations of the cutaneous lesions. In the patients visited to hospital with leishmaniasis suspicion, 65% (26/40) lesions were dry type, while 35% (14/40) were moist with central invaginations. Within the dry type, 46.15% (12/26) lesions had scales or crusted, 19.2% (5/26) had bulged appearance or nodulated and remaining had swollen erythematic appearance ( Table 2). Most of the lesions 77.5% (31/40) were localized while 22.5% (9/40) were dispersed lesions. Similarly, most of the lesions 60% (24/40) had single lesion while www.nature.com/scientificreports/ www.nature.com/scientificreports/ 40% (16/40) had multiple lesions. The lesions in the suspected cases were in the different exposed part of the body, mostly in the face (n = 19, 47.5%), followed by hand (n = 5, 12.5%), neck (n = 5, 12.5%), leg (n = 3, 7.5%), and abdominal part (n = 3, 7.5%) ( Table 2). Others having multiple lesions on these parts in the five patients (n = 5, 12.5%) (Supplementary Table S Observation of amastigote in smear and promastigote in culture. Among  Molecular diagnosis of cutaneous leishmaniasis and identification of causative species by minicircle kDNA PCR. The nested minicircle kDNA PCR, run along with negative control, detected 55% (22/40 suspected cases) to be PCR-positive cutaneous leishmaniasis cases. The amplicon sizes ranged from 700 to 800 bp in PCR-1 products whenever band was seen and the two bands observed in PCR-2 ( Fig. 4A1,A2, Table 3) confirmed the cutaneous leishmaniasis in the suspected cases. The two bands of PCR-2 indicated different Leishmania species circulating among the study population. Further, the amplicons of 600 bp and 720 bp confirmed etiologic species of L. major and L. donovani, respectively. There were 9 cases of cutaneous leishmaniasis caused due to the species L.major and 13 cases were due to L. donovani.   It has been noted that the L. donovani sequences were not grouped into a significant monophyletic cluster (only 72% bootstrap value) whereas the L. donovani plus L. infantum cluster was significantly monophyletic (96% bootstrap value). This indicates that the ITS1 marker is probably not sufficient to separate these extremely closely related species.

Discussion
In recent years, cutaneous leishmaniasis has emerged as an indigenous disease in Nepal, with increasing cases every year 11 . Moreover, lack of specialized knowledge causes medical practitioners to misdiagnose cases of cutaneous leishmaniasis as lupus vulgaris (well-defined skin-colored to erythematous plaque) 14,15 , basal cell carcinoma (nodular or nodulo-ulcerative lesions) 12 and leprosy, which is characterized by discolored (pale or reddish) and numb patch of skin 16 . So, the patients who visited the hospitals with complaints were previously treated without a confirmed diagnosis for leishmaniasis or other skin conditions. It has been noted that some drugs are effective for treating L. major 17,18 , which may have hide the leishmaniasis cases. Cutaneous lesions can either be a single, limited skin lesion or multiple, large, locally destructive skin lesions.
Leishmaniasis is diagnosed in many endemic areas based on clinical symptoms supported by a diagnostic test, such as the rK39 fast diagnostic test, and microscopic amastigotes observation, which requires a highly qualified technical individual. However, the reference microscopic diagnostic has a limited sensitivity and frequently returns false negative results 19 . The amplification of the parasite's kDNA gene has been reported as the most sensitive diagnostic test (98.7%) for the diagnosis of leishmaniasis 6,20 . However Nepal did not use PCR as a diagnostic tool until very recently that why the causative protozoan parasites of Nepalese cutaneous leishmaniasis www.nature.com/scientificreports/ cases remained unknown till 2018 before Prof. Manandhar's group detected L. major and L. donovani for the first time using PCR-based molecular diagnosis and species identification 15 . Species identification was the major aim of the present study to evaluate and identify the L. donovani persistence causing cutaneous leishmaniasis in Nepalese context, which have been done by PCR, restriction fragment length polymorphism (RFLP) and sequencing. The study made it possible to carry out minicircle kDNA based nested PCR test as the confirmatory diagnostic tool in Nepal for medically suspected microscopically negative cases. In this study, from skin lesion fine-needle aspirations, Leishmania kDNA PCR detected 22 positive cases (55%) out of 40 suspected patients being able to sort out symptomatically similar non-leishmaniasis cases. All the tools were able to discriminate Leishmania species circulating in Nepal. The 22 kDNA PCR positive samples were identified either as L. donovani (n = 13) or L major (n = 9). It showed that atypical L. donovani parasites was dominant with 59.1% to cause cutaneous leishmaniasis in Nepal over the typical etiological agent as L. major. The diagnostic and species identification tests were reconfirmed by ITS1 PCR which could detect 54.5% (n = 12/22) only and among them 5 cases as L. major out of 9 confirmed positive by kDNA PCR skipping the other 4 as undetected. Similarly, only the 7 cases of L donovani could be confirmed by ITS1 PCR among the 13 detected by kDNA ( Table 3). The sensitivity of the kDNA PCR is higher than that of ITS1 PCR as the copy number of the kDNA target is about 10,000 copies per cell 20 which is higher than ITS1 target regions having approximately 20-400 copies only 21 . So, kDNA PCR can detect DNA equivalent to < 0.001 parasites per reaction while the ITS1 PCR detects DNA equivalent to 0.2 parasites per reaction 6 . During the present study, parasitological diagnosis only confirmed twelve cases of cutaneous leishmaniasis similar to molecular diagnosis by ITS1 PCR among the 22 kDNA PCR positive cases. As already described by Bensoussan et al. 6 , diagnosis by kDNA PCR shows a higher sensitivity compared to parasitological diagnosis or ITS1 PCR. Therefore, the present study recommends kDNA PCR as an additional diagnostic technique, as also suggested by Pezeshkpoor et al. 22 .
The species identification were further confirmed by RFLP of the ITS1 gene amplicon which also clearly discriminated the patterns for L donovani and L. major as by Kumar et al. 12 (Fig. 4B2). The pattern showed similarity with that of the reference strain LEM 138 L. donovani parasite. Species identification is a vital part of the diagnostic procedure, especially in areas where more than one species of Leishmania circulate 23 . Clinical manifestation of cutaneous leishmaniasis is determined not only by the infecting species but also by the individual immune response 24 . Therefore, species determination simply by observing the clinical presentation may not be enough. The presence of multiple species of Leishmania in a region demands the molecular identification to help for the determination of the clinical prognosis and select the most appropriate regimen to be administered to each individual 25 .
The amplified ITS1 PCR products were processed to the sequencing on seven samples (Four of L. donovani and three of L. major). The L. donovani sequences which were not grouped into a significant monophyletic cluster (only 62% bootstrap value) had significantly monophyletic (99% bootstrap value) among the L. donovani and L. infantum cluster. This distinction is the indication that the ITS1 marker is probably not sufficient to separate these extremely closely related species. Lower bootstrap value on the phylogenetic analysis with indication of paraphyletic cluster and 99.47% homology with L. donovani from the non-endemic state in southern India (Gen-Bank Accession Number: MW053328.1) sequences is suggestive that the causative L. donovani species might be a new variant. So, further molecular analysis of these clinical isolates is needed to determine genetic makeup of the emerging atypical L. donovani variants. Our findings on L. donovani as the causative agent of cutaneous leishmaniasis is similar to other countries in Indian subcontinent like in Srilanka 26 and Bhutan 27 and India 28 . It would be an interesting study whether the L. donovani causing cutaneous leishmaniasis are of monophylectic www.nature.com/scientificreports/ cluster to the reported Indian subcontinent one. The information would be an interesting study for the other parts of the world as well where cutaneous leishmaniasis is gradually spreading to non-foci tropical and subtropical zones from temperate zone as observed in India 29 .
In this study, unexpectedly more cases of cutaneous leishmaniasis were detected in non-foci regions. Infections were seen in 16 districts encompassing all the seven provinces of Nepal spreading to all geographical Terai, Hill and Mountain regions. Interestingly, the higher cases were from hill (59%, 7 cases due to L. donovani and 6 by L. major) and mountain (32%; 6 cases due to L. donovani and 1 by L. major) regions situated at the latitude ranging from 27° 18′ 35.748′′ to 29° 59′ 12.192′′. The regions are in fact unusual foci for leishmaniasis and not yet identified as endemic regions by Kala Azar Elimination Program of Nepal Government for visceral leishmaniasis. www.nature.com/scientificreports/ Since country has not noted cutaneous leishmaniasis burden it has yet to realize those regions as potential endemic spots. Surprisingly, the least cases were detected from the overwhelmingly presumed Terai region (9%, 2 cases due to L.major). The infection in this region is further concerned issue as the causative species are only L. major despite the Terai region is endemic to visceral leishmaniasis caused by L donovani. The study results with confirmed cases due to L major (n = 9, 40.9%) and L donovani (n = 13, 59.1%) clearly showed that L donovani is the main etiological agent of cutaneous leishmaniasis in Nepal. Together the atypical cutaneous infections by L donovani parasite together with typical cutaneous infections by L major could be the reason for the increase of cutaneous leishmaniasis cases in Nepal. A larger and more in-depth study on cutaneous leishmaniasis may provide a broader picture of infection throughout Nepal. The increase in the number of cases of cutaneous leishmaniasis and the spread of the disease to the high altitudes of the country could be due to multiple factors like genetic drift in parasites, shift of biological cell tropism, human mobility, climate change and vector's susceptibility for hosting multiple species. This study provides some answers. Among the 13 positive cases of cutaneous leishmaniasis caused by L. donovani, only one (CL27) had travel history to endemic country outside Nepal (United Arab Emirates) and none of the other positive cases caused by L. donovani had travelled to visceral leishmaniasis endemic places either inside or outside Nepal. This non-travel history strongly supports the higher chances of genetic drift within the circulating L donovani in Nepal making it more adaptive for skin tissue tropism. The ITS1 sequencing data and the phylogenetic analysis (paraphyletic cluster with L donovani having only 62% bootstrap value) has also backed up illustratively indicating the possibility of a new L. donovani variant which might have been emerged with a new manifestation of cutaneous disease in Nepal. However, the whole genome sequencing of the circulating parasites could provide more answers to contribute to a better understanding of this L. donovani evolution, occurrence of Leishmania species hybrids (as L. major/L. donovani or L. donovani/L. donovani) and/or adaptation to its new tissue tropism [30][31][32][33][34] .
It also seems that L donovani is an opportunistic parasite which uses the tissue tropism selecting the skin cells for better adaptation to cause cutaneous leishmaniasis. The three infected mountain districts which were tagged as doubtful districts of visceral leishmaniasis by the Nepal Government validate the opportunistic nature of the parasites. The infection response also suggests that L donovani has been indigenously circulating even in the cold mountain region with better adoption 35 . At the verge of visceral leishmaniasis elimination as public health problem in Nepal in the 18 endemic districts 36 , the opportunistic tissue tropism of Leishmania species remains an unresolved question. It implicates numerous factors requiring an understanding the genetic determinants and the biology of tropism with the consideration of molecular and cellular factors of host-parasite-vector interactions 34,37,38 . These cutaneous leishmaniasis cases due to L. donovani are attention-grabbing and should be considered as potential reservoirs that could unknowingly enhance the continuation of parasite transmission and a risk of resurgence of visceral leishmaniasis 36 . This study found that there is no human reservoir since the family and the neighbor of the infected patient have neither visceral leishmaniasis nor cutaneous leishmaniasis and post-kala-azar dermal leishmaniasis. There might be the animal reservoir responsible for the transmission of the parasite. In Nepal, domestic animals such as cows, buffalo, and goats as well as the stray dog can be reservoir host for Leishmania spp. 39,40 . Eight districts which were not targeted for Kala azar elimination have also been found infected with clear indication that there is need of urgent surveillance integrating in the campaigns of the surveillance of visceral leishmaniasis.
In recent past, the road facilities in Nepal have been notably increased making the travel easy and swift from endemic areas. The climate change due to global warming has shown direct effect in the rise in atmospheric temperature favoring the survival of the thermophilic vector in the higher altitudes 15,41,42 and adopting the vectors for hosting multiple parasite species 43 . While analyzing on the potential transmission dimension due to travelling in endemic in-country and outside-country, it was detected that two cases (CL36 and CL39) had the travel history to the leishmaniasis endemic country (Saudi Arabia) where the subject had infection before returning to Nepal. Another case (CL-13) had travel history to hilly district, Darchula which is again categorized as doubtful endemic region. Both the cases were caused by L. major which is new species for Terai region where L donovani use to be pre-existing species causing visceral leishmaniasis. Hence, the study concludes that the L. major transmission in the Terai is via travel route of endemic regions. It indicates that Terai is yet not endemic to the cutaneous leishmaniasis but now onwards there is huge threat of quick indigenous circulation due to its favorable climate.
Genderwise disribution showed 59.09% (n = 13) male while 40.9% (n = 9) were female, most of the males were from the age group (21-40 yrs) and (41-60 yrs) which covers working age. In Nepal, females are mostly involved in household works and males are involved in outdoor works. This may have predisposed the males to the bite of sand fly. However, The gender-wise demography has not shown gender wise disparity (P = 0.666). The distribution of the disease in different age group was statistically not significant (P = 0.833), however age-wise prevalence showed that children-teenagers (< 20 yrs) and adult population (21-40 yrs) were most victimized. Since, the disease has been well known to cause skin lesions, mainly ulcers, leaving life-long scars and serious disability or psychosocial burden 44 . The present study showed morphology of the lesions showed that the most of the cases were localized (77.5%) and other were dispersed (22.5%), with 47.5% of the subjects affected in the facial part of the body followed by other exposed parts such as arms and neck. Therefore, the health issue should be addressed timely by diagnosing the cases promptly and appropriately before the localized lesions become dispersed and cases become severe.
Recently, World Health Organization (WHO) has published a new strategic framework for skin-related neglected tropical diseases to prevent, control, eliminate and eradicate of these diseases globally by 2030 including cutaneous leishmaniasis 3 . In the context of cutaneous leishmaniasis in Asia, the disease is present but uncommon 45 as in Nepal. The progression of cutaneous leishmaniasis cases and geographic expansion of areas where both L. major and L. donovani is circulating must be taken into consideration. There would be many folds of population who might have been infected but could not reach to health centers for checkup due to different reasons. So, the percentile infection of this study may not represent the actual infection dimension www.nature.com/scientificreports/ in the country. It needs an active surveillance implemented which helps for actual epidemiological map. The Infectious and Viral Disease Research Laboratory (IVDRL) of Tribhuvan University of Nepal, which successfully adopted the molecular PCR-based test for diagnosis of cutaneous leishmaniasis in Nepal for the first time, could be exploited for diagnosis as well as for surveillance of cutaneous leishmaniasis all over the country using all the PCR laboratory set-ups during the Covid-19 pandemics. Country may develop centers for both the visceral and cutaneous leishmaniasis surveillance and control program in every district or province since, L. donovani is endemic in 18 districts and 50 districts listed in doubtful endemic ones 11 , and cutaneous leishmaniasis is seen in 16 districts of all provinces. Further, cutaneous leishmaniasis disease and vector control programs should be launched at the earliest along with visceral leishmaniasis elimination program in order to implement leishmaniasis control management strategies as a whole. Accurate diagnostic tests should be implemented to detect and identify the causative Leishmania species of the disease, to help to avoid the misdiagnosis, to help clinicians to adapt suitable treatment, and update the distribution map of Leishmania species. To meet these needs, molecular diagnostic tools must be used for the diagnosis, understanding the emergence of new hotspots, control of reservoirs and Leishmania transmission. Hence, we strongly recommend the concerned health authorities to keep this validated kDNA minicircle PCR-based test in regular routine diagnosis of leishmaniasis for controlling the new emerging cutaneous disease in Nepal.
In conclusion, Nepal has a new threat of emerging cutaneous leishmaniasis with the concomitant presence of atypical skin infections due to L. donovani and typical due to L. major. The detected L. donovani species which do not show the monophyletic cluster with previously sequenced data (those having highest percentage identity and sequence similarity) indicated the need of mitochondrial and nuclear gene or whole genome sequencing to identify the possible different lineage of L. donovani circulating in Nepal. The transmission dynamics to the non-foci mountain and hill region is a serious concern not only for the Nepal but also for the whole World. Therefore, cutaneous leishmaniasis control program with kDNA based PCR diagnostic approach has to be commenced at the earliest.

Methods
Study area, patients and samples. The present study was conducted in Nepal between 2018 and 2020.
Patients with cutaneous lesions presented to the three government hospitals, namely Sukraraj Tropical and Infectious Disease Hospital (STIDH)-Kathmandu, Bir Hospital-Kathmandu and Nepalganj Medical College and Teaching Hospital-Nepalganj, were taken care of for a clinical diagnosis and biopsies of the skin lesions were taken for routine diagnostic analyses. In this passive case-finding, the skin lesions were cleaned with 70% alcohol and 100 µL of normal saline injected to the lesion area or in some cases, slit skin was done using a sterile scalpel followed by tissue aspiration from periphery of each nodule/papule. For this study, 40 patients presented with cutaneous lesions suspected for cutaneous leishmaniasis were included. The tissue samples were used for microscopic examination, culture and molecular characterization.
Microscopic examination and parasite culture. A small drop of undiluted tissue aspiration sample was used to prepare thin smear slides on the clean and grease free glass slides. The smear was left to air dry at room temperature. Fixation was done using 100% methanol for 30 s and rinsed with water followed by staining with 10% Giemsa for 30 min, rinsed off with tap water and allowed to get dry at room temperature. The slides were examined for amastigotes [Leishman-Donovan (LD) bodies] under 100X oil immersion in light microscope.
For culture, one to two drops lesion aspirates from each patient were inoculated to NNN tube aseptically. The cultured tubes were kept in BOD/cooling incubator at constant 26 °C for 9 days. The tubes were observed for growth of parasites from day-6 onwards in inverted microscope. After incubation period, the liquid media (~ 200 µL) of the culture having growth of parasites was inoculated to 25 cm 2 culture flask containing 5 mL M199 complete media with 10% FBS and 0.5% streptomycin and penicillin (Gibco).

DNA extraction and PCR amplification. DNA from each clinical tissue sample was extracted using
Quick-DNA™ Miniprep Plus Kit (Zymo research), following the manufacturer's instructions. By using the same method, DNA were also extracted from cultured parasites. The extracted DNA were kept at -20 °C until PCR analysis.
Two PCR assays were performed. For the first PCR assay, a kinetoplast minicircle DNA (kDNA) nested PCR was performed using CSB2F (C/GA/GTA/GCA GAA AC/TCC CGT TCA) and CSB1R (ATT TTT CG/CGA/TTTT/ CGC AGA ACG) primers for first round PCR (PCR-1), and 13Z (TCG CAG AAC GCC CCT) and LiR (ACT GGG GGT TGG TGT AAA ATAG) primers for second round PCR (PCR-2) as described by Noyes et al. 20 . Briefly, the PCR-1 consists of 94 °C for 2 min, followed by 40 cycles of amplification (94 °C for 50 s, 54 °C for 60 s, 72 °C for 90 s), and final extension at 72 °C for 10 min. In the PCR-2, 1 µL diluted PCR-1 product was used as template and same PCR conditions of PCR-1 were used, except annealing temperature which was increased from 54 to 56 °C. PCR amplicons were analyzed on 1.5% agarose gels. The 100 bp DNA ladder (Solis Biodyne) was used as the DNA molecular marker.
From DNA samples with a positive minicircle kDNA PCR result (from the second round PCR assay), an internal transcribed spacer-1 (ITS1) PCR was performed using LITSR (CTG GAT CAT TTT CCG ATG ) and L5.8S (TGA TAC CAC TTA TCG CAC ) primers to amplify the ribosomal ITS region as described by Kumar et al. 12  Sequencing and phylogenetic analysis. From seven PCR-confirmed cutaneous leishmaniasis patients, the ITS1 PCR products were purified using ExoSAP-IT Express (Affymetrix, Inc., CA, USA) according to the manufacturer's manual. Briefly, 5 µL of PCR product and 2 µL of Exosap was mixed and incubated at 37 °C for 10 min and then at 80 °C for 10 min. The purified product was subjected to sequencing reactions using BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) following the manufacturer instructions. The Big Dye XterminatorTM Kit (Applied Biosystems) was used to further purify these single-stranded sequences. Finally, the purified amplicons were sequenced in an automated 3500XL Genetic Analyzer (Applied Biosystems) 46 . The quality of raw sequences was checked in sequencer v. 5.0 (GeneCodes Corporation, MI, USA) through base calling and sequences were generated by using both strands consensus. For the present study, the ITS1 DNA sequences of the Leishmania spp parasites isolated from the seven PCR-confirmed cutaneous leishmaniasis patients were uploaded to National Center for Biotechnology Information (NCBI) and obtained respective accession numbers. For the identification of Leishmania spp., the final sequence obtained from each sample was subjected to Nucleotide Basic Local Alignment Search Tool (BLAST) to search similarity with the Leishmania spp. sequences stored on NCBI database (Shrestha et al. 46 ). Sequence alignment was performed by using the algorithm ClustalW; a Maximum Likelihood tree (ML tree) was constructed using the best model of multiple substitutions (i.e. the one presenting the Bayesian Information Criterion score (BIC score), and a statistical phylogeny test based on a bootstrap of 50,000 replicates was applied. The construction of ML tree was made with the software IQ-TREE v.2.2.0 47 while alignment and tree editing were achieve by the MEGA7 48 .
Statistical analysis and ethical statement. The data were statistically analyzed using Graph Pad Prism version 9.3.0 and SPSS version 21. Ethical approval was taken from Nepal Health Research Council. Written Informed Consent was taken from each patient informing the aim of the research, type of the study and method of sample collection before the enrollment in this research. In the case of minors, consents were given by their parents or guardians.
Ethical approval. The ethical approval for the research involving human participants was granted from Nepal Health Research Council (NHRC) (Reg. No. 45/2018 and 114/2019). All the methods were carried out in accordance with NHRC guidelines and regulations. Informed consent was obtained from all participants and/or their legal guardians for publication of their anonymized demographic and clinical information, and laboratory findings.